Nucleic acid encoding a signal mediator protein that induces cellular morphological alterations

ABSTRACT

An isolated nucleic acid molecule is provided which encodes a mammalian signal mediator protein involved in regulation of cellular morphological alterations. The encoded protein comprises an amino-terminal SH3 domain, an internal domain containing several SH2 binding motifs, and a carboxy-terminal effector domain that can induce pseudohyphal budding in yeast. The invention also provides the novel signal mediator protein, and antibodies thereto. These biological molecules are useful as research tools and as diagnostic and therapeutic agents for the identification, detection and regulation of complex signaling events leading to morphological, potentially neoplastic, cellular changes.

Pursuant to 35 U.S.C. §202(c), it is hereby acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the National Institutes of Health.

FIELD OF THE INVENTION

This invention relates to diagnosis and treatment of neoplastic diseases. More specifically, this invention provides novel nucleic acid molecules, proteins and antibodies useful for detection and/or regulation of complex signalling events leading to morphological and potentially neoplastic cellular changes.

BACKGROUND OF THE INVENTION

Cellular transformation during the development of cancer involves multiple alterations in the normal pattern of cell growth regulation. Primary events in the process of carcinogenesis involve the activation of oncogene function by some means (e.g., amplification, mutation, chromosomal rearrangement), and in many cases the removal of anti-oncogene function. In the most malignant and untreatable tumors, normal restraints on cell growth are completely lost as transformed cells escape from their primary sites and metastasize to other locations in the body. One reason for the enhanced growth and invasive properties of some tumors may be the acquisition of increasing numbers of mutations in oncogenes, with cumulative effect (Bear etal., Proc. Natl. Acad. Sci. USA 86:7495-7499, 1989). Alternatively, insofar as oncogenes function through the normal cellular signalling pathways required for organismal growth (reviewed in McCormick, Nature 363:15-16, 1993), additional events corresponding to mutations or deregulation in the oncogenic signalling pathways may also contribute to tumor malignancy (Gilks et al., Mol. Cell Biol. 13:1759-1768, 1993), even though mutations in the signalling pathways alone may not cause cancer.

Several discrete classes of proteins are known to be involved in conferring the different types of changes in cell division properties and morphology associated with transformation. These changes can be summarized as, first, the promotion of continuous cell cycling (immortalization); second, the loss of responsiveness to growth inhibitory signals and cell apoptotic signals; and third, the morphological restructuring of cells to enhance invasive properties.

Of these varied mechanisms of oncogene action, the role of control of cell morphology is one of the least understood. Work using non-transformed mammalian cells in culture has demonstrated that simply altering the shape of a cell can profoundly alter its pattern of response to growth signals (DiPersio et al., Mol. Cell Biol. 11:4405-4414, 1991), implying that control of cell shape may actually be causative of, rather than correlative to, cell transformation. For example, mutation of the antioncogene NF2 leads to development of nervous system tumors. Higher eucaryotic proteins involved in promoting aberrant morphological changes related to cancer may mediate additional functions in normal cells that are not obviously related to the role they play in cancer progression, complicating their identification and characterization. Identification and characterization of such genes and their encoded proteins would be beneficial for the development of therapeutic strategies in the treatment of malignancies.

Recent evidence suggests that certain key proteins involved in control of cellular morphology contain conserved domains referred to as SH2 and SH3 domains. These domains consist of non-catalytic stretches of approximately 50 amino acids (SH3) and 100 amino acids (SH2, also referred to as the "Src homology domain"). SH2/SH3 domains are found in cytoskeletal components, such as actin, and are also found in signalling proteins such as Abl. The interaction of these proteins may play a critical role in organizing cytoskeleton-membrane attachments.

Besides the numerous SH2/SH3 containing molecules with known catalytic or functional domains, there are several signalling molecules, called "adapter proteins," which are so small that no conserved domains seem to exist except SH2 and SH3 domains. Oncoproteins such as Nck, Grb2/Ash/SEM5 and Crk are representatives of this family. The SH2 regions of these oncoproteins bind specific phosphotyrosine-containing proteins by recognizing a phosphotyrosine in the context of several adjacent amino acids. Following recognition and binding, specific signals are transduced in a phosphorylation dependent manner.

As another example, P47v-Crk (CrK) is a transforming gene from avian sarcoma virus isolate CT10. This protein contains one SH2 and one SH3 domain, and induces an elevation of tyrosine phosphorylation on a variety of downstream targets. One of these targets, p130cas, is tightly associated with v-Crk. The SH2 domain of v-Crk is required for this association and subsequent cellular transformation. P130cas is also a substrate for Src mediated phosphorylation. Judging from its structure, p130cas may function as a "signal assembler" of Src family kinases and several cellular SH2-containing proteins. These proteins bind to the SH2 binding domain of p130cas, which is believed to induce a conformational change leading to the activation in inactivation of downstream signals, modulated by multiple domains of the protein.

Another oncogene, Ras, is a member of a large evolutionarily conserved superfamily of small GTP-binding proteins responsible for coordinating specific growth factor signals with specific changes in cell shape, including the development of stress fibers and membrane ruffles (Ridley and Hall, Cell 70:389-399, 1992; Ridley et al., Cell 70:401-410,1992). A rapidly growing family of oncoproteins, including Vav, Bcr, Ect-2, and Dbl, has been found to be involved in a variety of different tumors (Eva and Aaronson, Nature 316:273-275, 1985; Ron et al., EMBO J. 7:2465-2473, 1988; Adams et al., Oncogene 7:611-618, 1992; Miki et al., Nature 362:462-465, 1993). Proteins of this family have been shown to interact with Ras/Rac/Rho family members, and possess sequence characteristics that suggest they too directly associate with and modulate organization of the cytoskeleton.

In view of the significant relationship between signalling or "adapter" proteins, altered cellular morphology and the development of cancer, it would be of clear benefit to identify and isolate such proteins (or genes encoding them) for the purpose of developing diagnostic/therapeutic agents for the treatment of cancer. It is an object of the present invention to provide a purified nucleic acid molecule of mammalian origin that encodes a signal mediator protein (SMP) involved in the signalling cascade related to morphological cellular changes, and therefrom provide isolated and purified protein. Such a gene, when expressed in model systems, such as yeast, will provide utility as a research tool for identifying genes encoding interacting proteins in the signalling cascade. The gene may also be used diagnostically to identify related genes, and therapeutically in gene augmentation or replacement treatments. It is a further object of the present invention to provide derivatives of the SMP-encoding nucleic acid, such as various oligonucleotides and nucleic acid fragments for use as probes or reagents to analyze the expression of genes encoding the proteins. It is a further object of the invention to provide the signal mediator protein in purified form, and to provide antibodies immunologically specific for the signal mediator protein for the purpose of identifying and quantitating this mediator in selected cells and tissues.

SUMMARY OF THE INVENTION

This invention provides novel biological molecules useful for identification, detection and/or regulation of complex signalling events that regulate cellular morphological changes. According to one aspect of the present invention, an isolated nucleic acid molecule is provided that includes an open reading frame encoding a mammalian signal mediator protein of a size between about 795 and about 875 amino acids in length (preferably about 834 amino acids). The protein comprises an amino-terminal SH3 domain, an internal domain that includes a multiplicity of SH2 binding motifs, and a carboxy-terminal effector domain. When produced in Saccharomyces cerevisiae, the carboxy-terminal effector domain is capable of inducing pseudohyphal budding in the organism under predetermined culture conditions. In a preferred embodiment, an isolated nucleic acid molecule is provided that includes an open reading frame encoding a human mammalian signal mediator protein. In a particularly preferred embodiment, the human signal mediator protein has an amino acid sequence substantially the same as Sequence I.D. No. 2. An exemplary nucleic acid molecule of the invention comprises Sequence I.D. No. 1.

According to another aspect of the present invention, an isolated nucleic acid molecule is provided, which has a sequence selected from the group consisting of: (1) Sequence I.D. No. 1; (2) a sequence hybridizing with part or all of the complementary strand of Sequence I.D. No. 1 and encoding a polypeptide substantially the same as part or all of a polypeptide encoded by Sequence I.D. No. 1; and (3) a sequence encoding part or all of a polypeptide having amino acid Sequence I.D. No. 2.

According to another aspect of the present invention, an isolated nucleic acid molecule is provided which has a sequence that encodes a carboxy-terminal effector domain of a mammalian signal mediator protein. This domain has an amino acid sequence of greater than 74% similarity to the portion of Sequence I.D. No. 2 comprising amino acids 626-834.

According to another aspect of the present invention, an isolated mammalian signal mediator protein is provided which has a deduced molecular weight of between about 100 kDa and 115 kDa (preferably about 108 kDa). The protein comprises an amino-terminal SH3 domain, an internal domain that includes a multiplicity of SH2 binding motifs, and a carboxy-terminal effector domain, which is capable of inducing pseudohyphal budding in Saccharomyces cerevisiae under pre-determined culture conditions, as described in greater detail hereinbelow. In a preferred embodiment of the invention, the protein is of human origin, and has an amino acid sequence substantially the same as Sequence I.D. No. 2.

According to another aspect of the present invention, an isolated mammalian signal mediator protein is provided, which comprises a carboxy-terminal effector domain having an amino acid sequence of greater than 74% similarity to the portion of Sequence I.D. No. 2 comprising amino acids 626-834. In a preferred embodiment, the amino acid sequence of the carboxy-terminal effector domain is greater than about 50% identical to that portion of Sequence I.D. No. 2.

According to another aspect of the present invention, antibodies immunologically specific for the proteins described hereinabove are provided.

Various terms relating to the biological molecules of the present invention are used hereinabove and also throughout the specifications and claims. The terms "substantially the same," "percent similarity" and "percent identity (identical)" are defined in detail in the description set forth below.

With reference to nucleic acids of the invention, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it was derived. For example, the "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a procaryote or eucaryote.

With respect to RNA molecules of the invention, the term "isolated nucleic acid" primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a "substantially pure" form (the term "substantially pure" is defined below).

With respect to protein, the term "isolated protein" or "isolated and purified protein" is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure" form.

The term "substantially pure" refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

With respect to antibodies of the invention, the term "immunologically specific" refers to antibodies that bind to one or more epitopes of a protein of interest (e.g., SMP), but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

With respect to oligonucleotides, the term "specifically hybridizing" refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.

The nucleic acids, proteins and antibodies of the present invention are useful as research tools and will facilitate the elucidation of the mechanistic action of the novel genetic and protein interactions involved in the control of cellular morphology. They should also find broad utility as diagnostic and therapeutic agents for the detection and treatment of cancer and other proliferative diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Nucleotide sequence (Sequence I.D. No. 1) and deduced amino acid sequence (Sequence I.D. No. 2) of HEF1, a cDNA of human origin encoding an exemplary signal mediator protein of the invention.

FIG. 2. Amino acid sequence alignment of the deduced amino acid sequence of HEF1 (Sequence I.D. No. 2) with homologous sequences of p130cas from rat (Sequence I.D. No 3). Boxes represent regions of sequence identity between the two proteins. The closed circle marks the site of the initial methionine in the truncated clone of HEF1. The thick underline denotes the conserved SH3 domain. Tyrosines are marked with asterisks.

FIG. 3. Amino acid sequence alignment of the carboxy-terminal regions of HEF1-encoded hSMP with p130cas and the mouse homolog of hSMP, mSMP encoded by MEF1 (Sequence I.D. No. 4).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a novel gene has been isolated that encodes a protein involved in the signal transduction pathway that coordinates changes in cellular growth regulation. This protein is sometimes referred to herein as "signal mediator protein or "SMP."

Using a screen to identify human genes that promote psuedohyphal conversion in the yeast Saccharomyces cerevisiae, a 900 bp partial cDNA clone was obtained that causes strong pseudohyphal growth of S. cerevisiae on low nitrogen medium. This dimorphic shift from normal to "pseudohyphal" budding in yeast has been shown to involve the action of growth regulatory kinase cascades and cell cycle-related transcription factors (Gimeno & Fink, Mol. Cell Biol. 14: 2100-2112, 1994; Gimeno et al., Cell 68: 1077-1090, 992; Blacketer et al., Mol. Cell Biol. 13: 5567-5581, 1993; Liu et al. Science 262: 1741-1744, 1993).

Using the 900 bp partial cDNA clone as a probe in a combination of screening approaches, a full-length clone of approximately 3.7 kb was isolated. This clone encodes a single continuous open reading frame of about 834 amino acids, which constitutes the signal mediator protein of the invention. SMP is characterized by an amino-terminal SH3 domain and an adjacent domain containing multiple SH2 binding motifs. The protein also contains a carboxy terminal "effector" domain that is capable of inducing the shift to pseudo-hyphal budding in yeast. A cDNA encoding a mouse homolog of the carboxy-terminal "effector" region has also been identified (FIG. 3). Homology searches of the Genbank data base revealed an approximately 64% similarity on the amino acid level between SMP from human and the adapter protein, p130cas, recently cloned from rat (as disclosed by Sakai et al., EMBO J. 13: 3748-3756, 1994). However, p130cas is significantly larger than SMP (968 amino acids for rat p130cas versus 834 amino acids for human SMP), and differs with respect to amino acid composition. A comparison of SMP with p130cas is set forth in greater detail in Example 1.

The aforementioned human partial cDNA clone that enhanced pseudohyphal formation in yeast encodes only the carboxy-terminal portion of SMP, comprising about 182 amino acids. The enhancement of pseudohyphal formation by the carboxy-terminal fragment of SMP, in addition to the relatively high degree of homology with p130cas over this region, indicates that it is this domain that acts as an effector in regulating cellular morphology. Thus, this domain is sometimes referred to herein as a "C-terminal effector domain." It should be noted that, although the carboxy-terminal fragment of p130cas was also found capable of enhancing pseudohyphal formation, it did not do so to the same extent as the C-terminal domain of SMP (on a scale of 1 to 10, the SMP C-terminal domain is a "10," while the p130cas C-terminal domain is a "6"). The SMP C-terminal domain was also found to be involved in homodimerization and in heterodimerization with p130cas and, like p130cas, associates with Abl and appears to be phosphorylated by Abl.

Thus, SMP can be classified within a family of docking adapters, which includes p130cas, capable of multiple associations with signalling molecules and transduction of such signals to coordinate changes in cellular growth regulation. The SMP protein comprises, from amino- to carboxy-terminus, an SH3 domain, a poly-proline domain several SH2 binding motifs, a serine rich region, and the carboxy-terminal effector domain.

A human clone that encodes an exemplary signal mediator protein of the invention is sometimes referred to herein as "HEF1" (human enhancer of filamentation) to reflect the screening method by which it was in part identified. The nucleotide sequence of HEF1 is set forth herein as Sequence I.D. No. 1. The signal mediator protein encoded by HEF1 is sometimes referred to herein as hSMP. The amino acid sequence deduced from Sequence I.D. No. 1 is set forth herein as Sequence I.D. No. 2. The characteristics of human SMP are described in greater detail in Example 1.

It is believed that Sequence I.D. No. 1 constitutes a full-length SMP-encoding clone as it contains a suitable methionine for initiation of translation. This cDNA is approximately 3.7 kb in length. Northern analysis of a human multi-tissue RNA blot (Clontech MTNI) suggests a full-length transcript of approximately 3.4 kb. A second transcript of approximately 5.4 kb was also observed, which may represent an alternative splice or initiation site.

Although the human SMP-encoding gene, HEF1, is described and exemplified herein, this invention is intended to encompass nucleic acid sequences and proteins from other species that are sufficiently similar to be used interchangeably with SMP-encoding nucleic acids and proteins for the research, diagnostic and therapeutic purposes described below. Because of the high degree of conservation of genes encoding specific signal transducers and related oncogenes, it will be appreciated by those skilled in the art that, even if the interspecies SMP homology is low, SMP-encoding nucleic acids and SMP proteins from a variety of mammalian species should possess a sufficient degree of homology with SMP so as to be interchangeably useful with SMP in such diagnostic and therapeutic applications. Accordingly, the present invention is drawn to mammalian SMP-encoding nucleic acids and SMP proteins, preferably to SMP of primate origin, and most preferably to SMP of human origin. Accordingly, when the terms "signal mediator protein" or "SMP" or "SMP-encoding nucleic acid" are used herein, they are intended to encompass mammalian SMP-encoding nucleic acids and SMPs falling within the confines of homology set forth below, of which hSMP, preferably encoded by HEF1, is an exemplary member.

Allelic variants and natural mutants of Sequence I.D. No. 1 are likely to exist within the human genome and within the genomes of other mammalian species. Because such variants are expected to possess certain differences in nucleotide and amino acid sequence, this invention provides an isolated nucleic acid molecule and an isolated SMP protein having at least about 50-60% (preferably 60-80%, most preferably over 80%) sequence homology in the coding region with the nucleotide sequence set forth as Sequence I.D. No. (and, preferably, specifically comprising the coding region of sequence I.D. No. 1), and the amino acid sequence of Sequence I.D. No. 2. Because of the natural sequence variation likely to exist among signal mediator proteins and nucleic acids encoding them, one skilled in the art would expect to find up to about 40-50% sequence variation, while still maintaining the unique properties of the SMP of the present invention. Such an expectation is due in part to the degeneracy of the genetic code, as well as to the known evolutionary success of conservative amino acid sequence variations, which do not appreciably alter the nature of the protein. Accordingly, such variants are considered substantially the same as one another and are included within the scope of the present invention.

For purposes of this invention, the term "substantially the same" refers to nucleic acid or amino acid sequences having sequence variation that do not materially affect the nature of the protein (i.e. the structure and/or biological activity of the protein). With particular reference to nucleic acid sequences, the term "substantially the same" is intended to refer to the coding region and to conserved sequences governing expression, and refers primarily to degenerate codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide. With reference to amino acid sequences, the term "substantially the same" refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function. The terms "percent identity" and "percent similarity" are also used herein in comparisons among amino acid sequences. These terms are intended to be defined as they are in the UWGCG sequence analysis program (Devereaux et al., Nucl. Acids Res. 12: 387-397, 1984), available from the University of Wisconsin.

The following description sets forth the general procedures involved in practicing the present invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. Unless otherwise specified, general cloning procedures, such as those set forth in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989) (hereinafter "Sambrook et al.") are used.

I. Preparation of SMP-Encoding Nucleic Acid Molecules, Signal Mediator Proteins and Antibodies Thereto

A. Nucleic Acid Molecules

Nucleic acid molecules encoding the SMPs of the invention may be prepared by two general methods: (1) They may be synthesized from appropriate nucleotide triphosphates, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art.

The availability of nucleotide sequence information, such as the full length cDNA having Sequence I.D. No. 1, enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC). Long, double-stranded polynucleotides, such as a DNA molecule of the present invention, must be synthesized in stages, due to the size limitations inherent in current oligonucleotide synthetic methods. Thus, for example, a 3.7 kb double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity. Complementary segments thus produced may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire 3.7 kb double-stranded molecule. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.

Nucleic acid sequences encoding SMP may be isolated from appropriate biological sources using methods known in the art. In a preferred embodiment, a cDNA clone is isolated from an expression library of human origin. In an alternative embodiment, human genomic clones encoding SMP may be isolated. Alternatively, cDNA or genomic clones encoding from other mammalian species may be obtained.

In accordance with the present invention, nucleic acids having the appropriate level sequence homology with the protein coding region of Sequence I.D. No. 1 may be identified by using hybridization and washing conditions of appropriate stringency. For example, hybridizations may be performed, according to the method of Sambrook et al., using a hybridization solution comprising: 5×SSC, 5×Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37°-42° C. for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2×SSC and 1% SDS; (2) 15 minutes at room temperature in 2×SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37° C. in 1×SSC and 1% SDS; (4) 2 hours at 42-65° in 1×SSC and 1% SDS, changing the solution every 30 minutes.

Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector. In a preferred embodiment, clones are maintained in plasmid cloning/expression vector, such as p-Bluescript (Stratagene, La Jolla, Calif.), which is propagated in a suitable E. coli host cell.

SMP-encoding nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded. Thus, this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention, such as selected segments of the cDNA having Sequence I.D. No. 1. Such oligonucleotides are useful as probes for detecting SMP genes in test samples of potentially malignant cells or tissues, e.g. by PCR amplification, or for the isolation of homologous regulators of morphological control.

B. Proteins

A full-length SMP of the present invention may be prepared in a variety of ways, according to known methods. The protein may be purified from appropriate sources, e.g., human or animal cultured cells or tissues, by immunoaffinity purification. However, this is not a preferred method due to the low amount of protein likely to be present in a given cell type at any time.

The availability of nucleic acids molecules encoding SMP enables production of the protein using in vitro expression methods known in the art. For example, a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such a pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocytes. In vitro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wis. or BRL, Rockville, Md.

Alternatively, according to a preferred embodiment, larger quantities of SMP may be produced by expression in a suitable procaryotic or eucaryotic system. For example, part or all of a DNA molecule, such as the cDNA having Sequence I.D. No. 1, may be inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli, or into a baculovirus vector for expression in an insect cell. Such vectors comprise the regulatory elements necessary for expression of the DNA in the bacterial host cell, positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences.

The SMP produced by gene expression in a recombinant procaryotic or eucyarotic system may be purified according to methods known in the art. In a preferred embodiment, a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium. If expression/secretion vectors are not used, an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein. Such methods are commonly used by skilled practitioners.

The signal mediator proteins of the invention, prepared by the aforementioned methods, may be analyzed according to standard procedures. For example, such proteins may be subjected to amino acid sequence analysis, according to known methods.

The present invention also provides antibodies capable of immunospecifically binding to proteins of the invention. Polyclonal antibodies directed toward SMP may be prepared according to standard methods. In a preferred embodiment, monoclonal antibodies are prepared, which react immunospecifically with various epitopes of SMP. Monoclonal antibodies may be prepared according to general methods of Kohler and Milstein, following standard protocols. Polyclonal or monoclonal antibodies that immunospecifically interact with SMP can be utilized for identifying and purifying such proteins. For example, antibodies may be utilized for affinity separation of proteins with which they immunospecifically interact. Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules. Other uses of anti-SMP antibodies are described below.

II. Uses of SMP-Encoding Nucleic Acids, Signal Mediator Proteins and Antibodies Thereto

Cellular signalling molecules have received a great deal of attention as potential prognostic indicators of neoplastic disease and as therapeutic agents to be used for a variety of purposes in cancer chemotherapy. As a signalling molecule that induces profound morphological changes, SMP and related proteins from other mammalian species promise to be particularly useful research tools, as well as diagnostic and therapeutic agents.

A. SMP-Encoding Nucleic Acids

SMP-encoding nucleic acids may be used for a variety of purposes in accordance with the present invention. SMP-encoding DNA, RNA, or fragments thereof may be used as probes to detect the presence of and/or expression of genes encoding SMP. Methods in which SMP-encoding nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR).

The SMP-encoding nucleic acids of the invention may also be utilized as probes to identify related genes either from humans or from other species. As is well known in the art, hybridization stringencies may be adjusted to allow hybridization of nucleic acid probes with complementary sequences of varying degrees of homology. Thus, SMP-encoding nucleic acids may be used to advantage to identify and characterize other genes of varying degrees of relation to SMP, thereby enabling further characterization the signalling cascade involved in the morphological control of different cell types. Additionally, they may be used to identify genes encoding proteins that interact with SMP (e.g., by the "interaction trap" technique), which should further accelerate elucidation of these cellular signalling mechanisms.

Nucleic acid molecules, or fragments thereof, encoding SMP may also be utilized to control the expression of SMP, thereby regulating the amount of protein available to participate in oncogenic signalling pathways. Alterations in the physiological amount of "adapter protein" may act synergistically with chemotherapeutic agents used to treat cancer. In one embodiment, the nucleic acid molecules of the invention may be used to decrease expression of SMP in a population of malignant cells, In this embodiment, SMP proteins would be unable to serve as substrate acceptors for phosphorylation events mediated by oncogenes thereby effectively abrogating the activation signal. In this embodiment, antisense oligonucleotides expression. The use of antisense oligonucleotides to decrease expression levels of a pre-determined gene is known in the art. In a preferred embodiment, such antisense oligonucleotides are modified in various ways to increase their stability and membrane permeability, so as to maximize their effective delivery to target cells in vitro and in vivo. Such modifications include the preparation of phosphorothioate or methylphosphonate derivatives, among many others, according to procedures known in the art.

In another embodiment, overexpression of SMP is induced in a target population of cells to generate an excess of signal adapter molecules. This excess allows SMP to serve as a phosphorylation "sink" for the kinase activity of transforming oncogenes. Overexpression of SMP could lead to alterations in the cytoskeleton which could then be monitored with immunofluorescence or any other standard technique known in the art. Alternatively, overexpression of SMP by this method may facilitate the isolation and characterization of other components involved in the protein-protein complex formation that occurs via the SH2 homology domains during signal transduction.

As described above, SMP-encoding nucleic acids are also used to advantage to produce large quantities of substantially pure SMP protein, or selected portions thereof. In a preferred embodiment, the C-terminal "effector domain" of SMP is produced by expression of a nucleic acid encoding the domain. The full-length protein or selected domain is thereafter used for various research, diagnostic and therapeutic purposes, as described below.

B. Signal Mediator Protein and Antibodies

Purified SMP, or fragments thereof, may be used to produce polyclonal or monoclonal antibodies which also may serve as sensitive detection reagents for the presence and accumulation of SMP (or complexes containing SMP) in cultured cells or tissues from living patients (the term "patients" refers to both humans and animals). Recombinant techniques enable expression of fusion proteins containing part or all of the SMP protein. The full length protein or fragments of the protein may be used to advantage to generate an array of monoclonal antibodies specific for various epitopes of the protein, thereby providing even greater sensitivity for detection of the protein in cells or tissue.

Polyclonal or monoclonal antibodies immunologically specific for SMP may be used in a variety of assays designed to detect and quantitate the protein, which may be useful for rendering a prognosis as to a malignant disease. Such assays include, but are not limited to: (1) flow cytometric analysis; (2) immunochemical localization in SMP in cultured cells or tissues; and (3) immunoblot analysis (e.g., dot blot, Western blot) of extracts from various cells and tissues. Additionally, as described above, anti-SMP can be used for purification of SMP (e.g., affinity column purification, immunoprecipitation).

Anti-SMP antibodies may also be utilized as therapeutic agents to block the normal functionality of SMP in a target cell population, such as a tumor. Thus, similar to the antisense oligonucleotides described above, anti-SMP antibodies may be delivered to a target cell population by methods known in the art (i.e. through various lipophilic carriers that enable delivery of the compound of interest to the target cell cytoplasm) where the antibodies may interact with intrinsic SMP to render it nonfunctional.

From the foregoing discussion, it can be seen that SMP-encoding nucleic acids and SMP proteins of the invention can be used to detect SMP gene expression and protein accumulation for purposes of assessing the genetic and protein interactions involved in the regulation of morphological control pathways of a cell or tissue sample. Aberrant morphological changes are often correlatable with metastatic cellular proliferation in various cancers, such as breast cancer. It is expected that these tools will be particularly useful for diagnosis and prognosis of human neoplastic disease. Potentially of greater significance, however, is the utility of SMP-encoding nucleic acids, proteins and antibodies as therapeutic agents to disrupt the signal transduction pathways mediated by activated oncogenes that result in aberrant morphological cellular alterations.

Although the compositions of the invention have been described with respect to human diagnostics and therapeutics, it will be apparent to one skilled in the art that these tools will also be useful in animal and cultured cell experimentation with respect to various malignancies and/or other conditions manifested by alterations in cellular morphology. As diagnostic agents they can be used to monitor the effectiveness of potential anti-cancer agents on signal transduction pathways mediated by oncogenic proteins in vitro, and/or the development of neoplasms or malignant diseases in animal model systems. As therapeutics, they can be used either alone or as adjuncts to other chemotherapeutic drugs in animal models and veterinary applications to improve the effectiveness of such anti-cancer agents.

The following Example is provided to describe the invention in further detail. This Example is intended to illustrate and not to limit the invention.

EXAMPLE 1 Isolation and Characterization of a Nucleic Acid Molecule Encoding Human SMP

In this Example, we describe the cloning of a cDNA molecule encoding human SMP. This cDNA is sometimes referred to herein as HEF1 for human enhancer of filamentation, because of its identification in the pseudohyphal screen. We also provide an analysis of the structure of the human SMP (hSMP) as predicted from the deduced amino acid sequence encoded by the cDNA. Additionally, we describe the antibodies immunospecific for the recombinant hSMP protein, and their use in immunological detection of phosphorylated SMP from normal and Abl transformed NIH3T3 cells.

Pseudohyphal Budding Assay

The pseudohyphal budding assay was performed essentially as described previously (Gimeno et al. (1992) Cell 68:1077-1090; Gimeno et al. (1993) Characterization of Saccharomyces cerevisiae pseudohyphal growth, p. 83-103. In H. Vanden Bossche et al., eds. dimorphic fungi in biology and medicine, Plenum, N.Y.) Trp+ transformants were plated onto nitrogen restricted media. SLAGR is identical to SLAD, but with 2% galactose, 1% raffinose as a carbon source. Following 1-3 days growth at 30° C., the cells were observed under a Wild dissecting microscope to assess alterations in cellular morphology. A yeast grown on standard media multiplies until it forms a visible structure which appears as a hemispherical colony with a smooth circular outline. This morphology is strikingly homogeneous, with little variation from colony to colony. Pseudohyphal growth results from a reiterated pattern of unipolar cell division to form the chain of cells that constitute a pseudohypha. In polarized colonies which resemble colonies formed by filamentous fungi, the pseudohyphae radiate outward in all directions.

Isolation of cDNA and cloning

A HeLa cDNA library constructed in the TRPl+vector JG4-4 (Gyuris et al., Cell 75:791-803(1993)), was translated with inserts expressed as native proteins under the control of the galactose-inducible GALl promoter, into CG×74 yeast (MATa/e trpl/trpl; see Gimeno et al., 1992, supra). TRP+ transformants were plated to the nitrogen-restricted SLAGR medium (like SLAD, but with 2% galactose, 1% raffinose as a carbon source), and 120,000 colonies were visually screened using a Wild dissecting microscope at 50× amplification to identify colonies that produced pseudohyphae more extensively than background. cDNAs from these colonies were isolated and retransformed to naive CG×74; those that reproducibly generated enhanced pseudohyphae were sequenced. A 900 bp cDNA encoding a 182 amino acid open reading frame corresponding to the COOH-terminus of hSMP (HEF1-Cterm 182) possessed the most dramatic phenotype of cDNA obtained in this screen. Using the original 900 bp cDNA isolated in the pseudohyphal screen to probe a placental cDNA library cloned in lambda gt11, a larger clone (3.4 kb) was isolated. The longer clone obtained in this screen was used as a basis for 5' RACE using a kit from Clontech containing RACE-ready cDNA prepared from human kidney. Three independent clones from the RACE approach yielded identical 5' end-points located 18 base pairs upstream of the ATG encoding the first methionine in the sequence shown in FIG. 1. Repeated efforts with multiple primer sets showed no evidence for an N-terminally extended sequence. The full length clone, HEF1, is about 3.7 kb and encodes a protein about 835 amino acids in length.

Sequence Analysis

Both strands of the HEF1 clone were sequenced using oligonucleotide primers to the JG4-4 vector and to internal HEF1 sequences in combination with the Sequenase system (United States Biochemical) Database searching was performed using the BLAST algorithm (Altschul et al., J. Mol. Biol. 215:403-410, 1990) and sequence analysis was carried out using the package of programs from UWGCG (Devereux et al., Nucl. Acids Res. 12:387-397, 1984).

Northern Analysis

HEF1 cDNA was labelled with ³² P-dCTP by random priming, and used to probe a Northern blot containing 2 μg/lane human mRNA from multiple tissues. The blot was stripped and reprobed with a ³² P-labelled oligonucleotide specific for actin as a control for equivalent loading.

Immunoprecipitation and Western Blotting

Immunoprecipitation of hSMP from normal and Abl transformed NIH 3T3 cells was accomplished using polyclonal antiserum raised against a peptide derived from the hSMP C-terminus. Immunoprecipitates were resolved by electrophoresis on a 12% SDS-polyacrylamide gel. Following electrophoresis, immunoprecipitates were transferred to nitrocellulose, and reprobed with anti-phosphotyrosine antibody (4G10).

Growth Profiles

Yeast were transformed with HEF1 or vector alone and grown to saturated overnight cultures in trp⁻ glucose defined minimal medium, and re-diluted to OD600 <0.05 in trp⁻ galactose for growth curves. Growth curves were performed, with readings taken at 90 minute intervals for 12 hours, and at less frequent intervals up to 48 hours or longer.

Interaction Trap or Two Hybrid Analysis

EGY48 yeast (Gyuris et al., 1993, supra) were transformed by standard methods with plasmids expressing LexA-fusions, activation-domain fusions, or both, together with the LexA operator-LacZ reporter SH18-34 (Gyuris et al., 1993, supra). For all fusion proteins, synthesis of a fusion protein of the correct length in yeast was confirmed by Western blot assays of yeast extracts (Samson et al., Cell 57: 1045-1052, 1989) using polyclonal antiserum specific for LexA (Brent and Ptashne, Nature 312: 612-615, 1984) or for hemagglutinin (Babco, Inc), as appropriate. Activation of the LacZ reporter was determined as previously described (Brent and Ptashne, Cell 43: 729-736, 1985). Beta-galactosidase assays were performed on three independent colonies, on three separate occasions, and values for particular plasmid combinations varied less than 25%. Activation of the LEU2 reporter was determined by observing the colony forming ability of yeast plated on complete minimal medium lacking leucine. The LexA-PRD/HD expressing plasmid has been described (Golemis and Brent, Mol. Cell Biol. 12: 3006-3014, 1992).

RESULTS

Overexpression of the C-terminal domain of SMP influences Saccharomyces cerevisiae cell morphology. To identify proteins that regulate the morphology and polarity of human cells, a human cDNA library was screened for genes which enhanced formation of pseudohyphae when expressed in S. cerevisiae. The yeast undergoes a dimorphic shift in response to severe nitrogen limitation that involves changes in budding pattern, cell cycle control, cell elongation, and invasive growth into agar (Gimeno etal., 1992, supra). A galacross-inducible HeLa cell cDNA library was used to transform a yeast strain that can form pseudohyphae on nitrogen-restricted media, and a number of human genes which specifically enhanced pseudohyphal formation were identified. One of the cDNAs derived from this screen was found to cause the constitutive formation of pseudohyphae on rich and nitrogen restricted media. This cDNA is sometimes referred to as "HEF1-Cterm182" (because it encodes 182 amino acids of the C-terminal domain of the human SMP). A full-length clone containing the cDNA sequence was thereafter obtained. Analysis of the sequence of this cDNA (Sequence I.D. No. 1; FIG. 1) revealed that it was a novel human gens with strong sequence similarity to the rat p130cas gens (as disclosed by Sakai et al. EMBO J. 13: 3748-3756, 1994). This gene was designated HEF1, and its encoded protein was designated hSMP (Sequence I.D. No. 2). A comparison of the amino acid compositions (% by weight) of the HEF1-encoded hSMP and the rat p130cas is shown in Table 1 below.

                  TABLE 1                                                          ______________________________________                                                        % Composition                                                   Amino Acid       hSMP    p130cas                                               ______________________________________                                         Alanine          4.3     6.2                                                   Arginine         6.1     7.5                                                   Asparagine       4.1     1.8                                                   Aspartic acid    5.6     6.5                                                   Cysteine         1.5     0.6                                                   Glutamine        8.3     8.1                                                   Glutamic acid    6.6     5.8                                                   Glycine          3.5     4.5                                                   Histidine        4.0     3.1                                                   Isoleucine       4.2     1.6                                                   Leucine          8.7     9.6                                                   Lysine           6.2     4.8                                                   Methionine       2.8     1.0                                                   Phenylalanine    3.2     1.6                                                   Proline          7.0     11.1                                                  Serine           6.6     6.7                                                   Threonine        4.8     4.9                                                   Tryptophan       1.1     1.1                                                   Tyrosine         4.8     4.7                                                   Valine           5.6     7.7                                                   ______________________________________                                    

The deduced length of HEF1-encoded hSMP is amino acids and its deduced molecular weight is about 107,897 Da. The deduced length of the rat p130cas is 968 amino acids and its deduced molecular weight is about 121,421 Da.

Tissue specific expression of HEF1. RNA production was assessed by Northern blot analysis. HEF1 is expressed as two predominant transcripts of approximately 3.4 and 5.4 kb. Although present in all tissues examined (heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas), these transcripts are present at significantly higher levels in kidney, lung, and placenta. In contrast, a more uniform distribution throughout the body has been reported for p130cas. Two other cross-hybridizing minor species were detected, migrating at 8.0 kb in lung and 1.2 kb in liver. These may represent alternatively spliced HEF1 transcripts or other HEF1/p130cas related genes. HEF1 represents a distinct gene from p130cas rather than a human homolog, inasmuch as a screen of a murine genomic library with HEF1 cDNA led to identification of an exon that encoded a mouse C-terminal effector protein having a sequence essentially identical to hSMP-Cterm182 (FIG. 3). Furthermore, probe of a zoo blot at high stringency with a HEF1 cDNA probe indicates this gene is highly conserved from humans to yeast.

hSMP does not induce constitutive pseudohyphal budding by causing severe cell stress. The possibility that the C-terminal domain of hSMP was enhancing pseudohyphae formation by causing severe cell stress was excluded by comparing the growth rates of yeast containing the HEF1-cterm182 cDNA to yeast containing the expression vector control on plates and in liquid culture, with galactose as a sugar source to induce expression of HEF1-cterm182. The growth rate data shows that SMP-encoding genes are not simply toxic to yeast.

SMP belongs to a class of "adapter proteins" important in signalling cascades influencing morphological control. The HEF1 gene is approximately 3.7 kb and encodes a single continuous open reading frame of about 835 amino acids. The predicted hSMP protein notably contains an amino-terminal SH3 domain and an adjacent domain containing multiple SH2 binding motifs. Homology search of the Genbank database revealed that hSMP is 64% similar at the amino acid level to the adapter protein p130cas, recently cloned from rat (Sakai et al., EMBO J. 13:3748-3756, 1994). The amino acid alignment of hSMP and p130cas is shown in FIG. 2. P130cas was determined to be the predominant phosphorylated species in cells following transformation by the oncoprotein Crk and also complexes with, and is a substrate for Abl and Src. As shown in Table 2 below, the homology between SMP and p130cas is most pronounced over the SH3 domain (92% similarity, 74% identity) and in the region corresponding to the SMP-Cterm182 fragment (74% similarity, 57% identity). Although the domain containing SH2-binding motifs is more divergent from p130cas, SMP similarly possesses a large number of tyrosines in this region. The majority of SH2 binding sites in p130cas match the consensus for the SH2 domain of the oncoprotein Crk, while the amino acids flanking the tyrosine residues in SMP are more diverse, suggesting a broader range of associating proteins. Various SH2 binding motifs conserved between hSMP and p130cas are shown in Table 3.

                  TABLE 2                                                          ______________________________________                                         Domain Alignment: hSMP and p130cas                                             (Domains from amino to carboxyl terminus down the Table)                                 Size (a.a.) % Similarity/Identity                                    Domain      hSMP    p130cas   (hSMP:p130cas)                                   ______________________________________                                         SH3          50      50       92% similar,                                                                   74% indentical                                   Polyproline  10      38       (not compared)                                   SH2 binding 290     410       55% similar,                                     motifs                        36% identical                                    Serine-rich 250     260       56% similar,                                     region                        35% identical                                    C-terminal  210     210       74% similar,                                     effector domain               57% identical                                    ______________________________________                                    

                  TABLE 3                                                          ______________________________________                                         Conserved SH2 Binding Motifs and Associating Proteins                          SH2 Binding Motif   Associating Proteins                                       ______________________________________                                         YDIP                                                                           YDVP                Crk                                                        YDFP                                                                           YEYP                Vav or fps/fes                                             YAIP                Ab1                                                        YQNQ                Grb2                                                       YQVP                                                                           YQKD                                                                           YVYE                Novel                                                      YPSR                                                                           YNCD                                                                           ______________________________________                                    

The enhancement of pseudohyphal formation by hSMP-Cterm182 fragment in addition to the relatively high degree of homology to p130cas suggests that this domain acts as an effector in regulating cellular morphology. A test was performed to assay whether the homologous region of p130cas also enhanced pseudohyphal formation. The results show that the C-terminal fragment of p130cas did enhance psuedohyphal formation but not to the same extent as the C-terminal fragment of SMP. SMP was found to induce the strongest pseudohyphal phenotype of all cDNA fragments tested. By comparison, p130cas and another pseudohyphal inducer, RBP7 (subunit 7 of human RNA polymerase II, Golemis et al., Mol. Biol. of the Cell, 1995, in press) were only about 60% as effective as the hSMP-Cterm182 fragment.

The possible functions for the novel carboxy-terminal domains were investigated further using two-hybrid analysis. These experiments revealed that this domain mediated SMP homodimerization, and SMP/p130cas heterodimerization, yet failed to interact with non-specific control proteins.

SMP is a substrate for oncogene mediated phosphorylation. SMP was immunoprecipitated from normal and v-Abl transformed NIH3T3 cells using polyclonal antisera raised against a MAP peptide derived from the hSMP C-terminal domain. Probe of these immunoprecipitates with antibody to phosphotyrosine revealed a species migrating at approximately 130-140 kD that was specifically observed in Abl-transformed fibroblasts. This species may represent SMP phosphorylated by Abl, as SMP possesses a good match to SH2 binding domain recognized by Abl. The larger apparent molecular weight as compared with hSMP deduced molecular weight may reflect glycosylation or may be a result of its phosphorylated state.

SMP dimerizes with other important cellular regulatory proteins. To assay whether SMP dimerizes with other cellular proteins, the interaction trap/two hybrid analysis system was used. Briefly, a LexA-fusion and an epitope-tagged, activation-domain fusion to SMP were synthesized. The expression of proteins of the predicted size in yeast was confirmed using antibodies specific for the fusion moieties. Using a LexA-operator reporter, it was observed that LexA-SMP fusion protein activates transcription extremely weakly. However, LexA-SMP is able to interact with co-expressed activation domain-fused SMP to activate transcription of the reporter, indicating that it is able to form dimers (or higher order multimers).

SMP joins p130cas in defining a new family of docking adapters that, through multiple associations with signalling molecules via SH2 binding domains, is likely to coordinate changes in cellular growth regulation. The interactions between SMP homodimers and SMP-p130cas heterodimers may negatively regulate SMP and p130cas proteins by making them inaccessible to their targets. Alternatively, SMP and p130cas could work together to recruit new proteins to the signalling complex. The fact that the novel C-terminal domain shared between SMP and p130cas has the ability to cause pseudohyphal formation in yeast suggests that these proteins may directly alter cellular morphology by interacting with the cytoskeleton. In fact, previous yeast-morphology based screens for higher eucaryotic proteins have tended to isolate cytoskeletally related proteins. This invention therefore provides reagents influencing the changes in cell morphology that accompany oncoprotein-mediated transformation in carcinogenesis.

The present invention is not limited to the embodiments specifically described above, but is capable of variation and modification without departure from the scope of the appended claims.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 4                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3672 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        ACCCCCACGCTACCGAAATGAAGTATAAGAATCTTATGGCAAGGGCCTTATATGACAATG60                 TCCCAGAGTGTGCCGAGGAACTGGCCTTTCGCAAGGGAGACATCCTGACCGTCATAGAGC120                AGAACACAGGGGGACTGGAAGGATGGTGGCTGTGCTCGTTACACGGTCGGCAAGGCATTG180                TCCCAGGCAACCGGGTGAAGCTTCTGATTGGCCCCATGCAGGAGACTGCCTCCAGTCACG240                AGCAGCCTGCCTCTGGACTGATGCAGCAGACCTTTGGCCAACAGAAGCTCTATCAAGTGC300                CAAACCCACAGGCTGCTCCCCGAGACACTATCTACCAAGTGCCACCTTCCTACCAAAATC360                AGGGAATTTACCAAGTCCCCACTGGCCACGGCACCCAAGAACAAGAGGTATATCAGGTGC420                CACCATCAGTGCAGAGAAGCATTGGGGGAACCAGTGGGCCCCACGTGGGTAAAAAGGTGA480                TAACCCCCGTGAGGACAGGCCATGGCTACGTATACGAGTACCCATCCAGATACCAAAAGG540                ATGTCTATGATATCCCTCCTTCTCATACCACTCAAGGGGTATACGACATCCCTCCCTCAT600                CAGCAAAAGGCCCTGTGTTTTCAGTTCCAGTGGGAGAGATAAAACCTCAAGGGGTGTATG660                ACATCCCGCCTACAAAAGGGGTATATGCCATTCCGCCCTCTGCTTGCCGGGATGAAGCAG720                GGCTTAGGGAAAAAGACTATGACTTCCCCCCTCCCATGAGACAAGCTGGAAGGCCGGACC780                TCAGACCGGAGGGGGTTTATGACATTCCTCCAACCTGCACCAAGCCAGCAGGGAAGGACC840                TTCATGTAAAATACAACTGTGACATTCCAGGAGCTGCAGAACCGGTGGCTCGAAGGCACC900                AGAGCCTGTCCCCGAATCACCCACCCCCGCAACTCGGACAGTCAGTGGGCTCTCAGAACG960                ACGCATATGATGTCCCCCGAGGCGTTCAGTTTCTTGAGCCACCAGCAGAAACCAGTGAGA1020               AAGCAAACCCCCAGGAAAGGGATGGTGTTTATGATGTCCCTCTGCATAACCCGCCAGATG1080               CTAAAGGCTCTCGGGACTTGGTGGATGGGATCAACCGATTGTCTTTCTCCAGTACAGGCA1140               GCACCCGGAGTAACATGTCCACGTCTTCCACCTCCTCCAAGGAGTCCTCACTGTCAGCCT1200               CCCCAGCTCAGGACAAAAGGCTCTTCCTGGATCCAGACACAGCTATTGAGAGACTTCAGC1260               GGCTCCAGCAGGCCCTTGAGATGGGTGTCTCCAGCCTAATGGCACTGGTCACTACCGACT1320               GGCGGTGTTACGGATATATGGAAAGACACATCAATGAAATACGCACAGCAGTGGACAAGG1380               TGGAGCTGTTCCTGAAGGAGTACCTCCACTTTGTCAAGGGAGCTGTTGCAAATGCTGCCT1440               GCCTCCCGGAACTCATCCTCCACAACAAGATGAAGCGGGAGCTGCAACGAGTCGAAGACT1500               CCCACCAGATCCTGAGTCAAACCAGCCATGACTTAAATGAGTGCAGCTGGTCCCTGAATA1560               TCTTGGCCATCAACAAGCCCCAGAACAAGTGTGACGATCTGGACCGGTTTGTGATGGTGG1620               CAAAGACGGTGCCCGATGACGCCAAGCAGCTCACCACAACCATCAACACCAACGCAGAGG1680               CCCTCTTCAGACCCGGCCCTGGCAGCTTGCATCTGAAGAATGGGCCGGAGAGCATCATGA1740               ACTCAACGGAGTACCCACACGGTGGCTCCCAGGGACAGCTGCTGCATCCTGGTGACCACA1800               AGGCCCAGGCCCACAACAAGGCACTGCCCCCAGGCCTGAGCAAGGAGCAGGCCCCTGACT1860               GTAGCAGCAGTGATGGTTCTGAGAGGAGCTGGATGGATGACTACGATTACGTCCACCTAC1920               AGGGTAAGGAGGAGTTTGAGAGGCAACAGAAAGAGCTATTGGAAAAAGAGAATATCATGA1980               AACAGAACAAGATGCAGCTGGAACATCATCAGCTGAGCCAGTTCCAGCTGTTGGAACAAG2040               AGATTACAAAGCCCGTGGAGAATGACATCTCGAAGTGGAAGCCCTCTCAGAGCCTACCCA2100               CCACAAACAGTGGCGTGAGTGCTCAGGATCGGCAGTTGCTGTGCTTCTACTATGACCAAT2160               GTGAGACCCATTTCATTTCCCTTCTCAACGCCATTGACGCACTCTTCAGTTGTGTCAGCT2220               CAGCCCAGCCCCCGCGAATCTTCGTGGCACACAGCAAGTTTGTCATCCTCAGTGCACACA2280               AACTGGTGTTCATTGGAGACACGCTGACACGGCAGGTGACTGCCCAGGACATTCGCAACA2340               AAGTCATGAACTCCAGCAACCAGCTCTGCGAGCAGCTCAAGACTATAGTCATGGCAACCA2400               AGATGGCCGCCCTCCATTACCCCAGCACCACGGCCCTGCAGGAAATGGTGCACCAAGTGA2460               CAGACCTTTCTAGAAATGCCCAGCTGTTCAAGCGCTCTTTGCTGGAGATGGCAACGTTCT2520               GAGAAGAAAAAAAAGAGGAAGGGGACTGCGTTAACGGTTACTAAGGAAAACTGGAAATAC2580               TGTCTGGTTTTTGTAAATGTTATCTATTTTTGTAGATAATTTTATATAAAAATGAAATAT2640               TTTAACATTTTATGGGTCAGACAACTTTCAGAAATTCAGGGAGCTGGAGAGGGAAATCTT2700               TTTTTCCCCCCTGAGTNGTTCTTATGTATACACAGAAGTATCTGAGACATAAACTGTACA2760               GAAAACTTGTCCACGTCCTTTTGTATGCCCATGTATTCATGTTTTTGTTTGTAGATGTTT2820               GTCTGATGCATTTCATTAAAAAAAAAACCATGAATTACGAAGCACCTTAGTAAGCACCTT2880               CTAATGCTGCATTTTTTTTGTTGTTGTTAAAAACATCCAGCTGGTTATAATATTGTTCTC2940               CACGTCCTTGTGATGATTCTGAGCCTGGCACTGGGAATCTGGGAAGCATAGTTTATTTGC3000               AAGTGTTCACCTTCCAAATCATGAGGCATAGCATGACTTATTCTTGTTTTGAAAACTCTT3060               TTCAAAACTGACCATCTTAAACACATGATGGCCAAGTGCCACAAAGCCCTCTTGCGGAGA3120               CATTTACGAATATATATGTGGATCCAAGTCTCGATAGTTAGGCGTTGGAGGGAAGAGAGA3180               CCAGAGAGTTTAGAGGCCAGGACCACAGTTAGGATTGGGTTGTTTCAATACTGAGAGACA3240               GCTACAATAAAAGGAGAGCAATTGCCTCCCTGGGGCTGTTCAATCTTCTGCATTTGTGAG3300               TGGTTCAGTCATGAGGTTTTCCAAAAGATGTTTTTAGAGTTGTAAAAACCATATTTGCAG3360               CAAAGATTTACAAAGGCGTATCAGACTATGATTGTTCACCAAAATAGGGGAATGGTTTGA3420               TCCGCCAGTTGCAAGTAGAGGCCTTTCTGACTCTTAATATTCACTTTGGTGCTACTACCC3480               CCATTACCTGAGGAACTGGCCAGGTCCTTGATCATGGAACTATAGAGCTACCAGACATAT3540               CCTGCTCTCTAAGGGAATTTATTGCTATCTTGCACCTTCTTTAAAACTCAAAAAACATAT3600               GCAGACCTGACACTCAAGAGTGGCTAGCTACACAGAGTCCATCTAATTTTTGCAACTTCC3660               CCCCCCGAATTC3672                                                               (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 834 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: Not Relevant                                                 (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetLysTyrLysAsnLeuMetAlaArgAlaLeuTyrAspAsnValPro                               151015                                                                         GluCysAlaGluGluLeuAlaPheArgLysGlyAspIleLeuThrVal                               202530                                                                         IleGluGlnAsnThrGlyGlyLeuGluGlyTrpTrpLeuCysSerLeu                               354045                                                                         HisGlyArgGlnGlyIleValProGlyAsnArgValLysLeuLeuIle                               505560                                                                         GlyProMetGlnGluThrAlaSerSerHisGluGlnProAlaSerGly                               65707580                                                                       LeuMetGlnGlnThrPheGlyGlnGlnLysLeuTyrGlnValProAsn                               859095                                                                         ProGlnAlaAlaProArgAspThrIleTyrGlnValProProSerTyr                               100105110                                                                      GlnAsnGlnGlyIleTyrGlnValProThrGlyHisGlyThrGlnGlu                               115120125                                                                      GlnGluValTyrGlnValProProSerValGlnArgSerIleGlyGly                               130135140                                                                      ThrSerGlyProHisValGlyLysLysValIleThrProValArgThr                               145150155160                                                                   GlyHisGlyTyrValTyrGluTyrProSerArgTyrGlnLysAspVal                               165170175                                                                      TyrAspIleProProSerHisThrThrGlnGlyValTyrAspIlePro                               180185190                                                                      ProSerSerAlaLysGlyProValPheSerValProValGlyGluIle                               195200205                                                                      LysProGlnGlyValTyrAspIleProProThrLysGlyValTyrAla                               210215220                                                                      IleProProSerAlaCysArgAspGluAlaGlyLeuArgGluLysAsp                               225230235240                                                                   TyrAspPheProProProMetArgGlnAlaGlyArgProAspLeuArg                               245250255                                                                      ProGluGlyValTyrAspIleProProThrCysThrLysProAlaGly                               260265270                                                                      LysAspLeuHisValLysTyrAsnCysAspIleProGlyAlaAlaGlu                               275280285                                                                      ProValAlaArgArgHisGlnSerLeuSerProAsnHisProProPro                               290295300                                                                      GlnLeuGlyGlnSerValGlySerGlnAsnAspAlaTyrAspValPro                               305310315320                                                                   ArgGlyValGlnPheLeuGluProProAlaGluThrSerGluLysAla                               325330335                                                                      AsnProGlnGluArgAspGlyValTyrAspValProLeuHisAsnPro                               340345350                                                                      ProAspAlaLysGlySerArgAspLeuValAspGlyIleAsnArgLeu                               355360365                                                                      SerPheSerSerThrGlySerThrArgSerAsnMetSerThrSerSer                               370375380                                                                      ThrSerSerLysGluSerSerLeuSerAlaSerProAlaGlnAspLys                               385390395400                                                                   ArgLeuPheLeuAspProAspThrAlaIleGluArgLeuGlnArgLeu                               405410415                                                                      GlnGlnAlaLeuGluMetGlyValSerSerLeuMetAlaLeuValThr                               420425430                                                                      ThrAspTrpArgCysTyrGlyTyrMetGluArgHisIleAsnGluIle                               435440445                                                                      ArgThrAlaValAspLysValGluLeuPheLeuLysGluTyrLeuHis                               450455460                                                                      PheValLysGlyAlaValAlaAsnAlaAlaCysLeuProGluLeuIle                               465470475480                                                                   LeuHisAsnLysMetLysArgGluLeuGlnArgValGluAspSerHis                               485490495                                                                      GlnIleLeuSerGlnThrSerHisAspLeuAsnGluCysSerTrpSer                               500505510                                                                      LeuAsnIleLeuAlaIleAsnLysProGlnAsnLysCysAspAspLeu                               515520525                                                                      AspArgPheValMetValAlaLysThrValProAspAspAlaLysGln                               530535540                                                                      LeuThrThrThrIleAsnThrAsnAlaGluAlaLeuPheArgProGly                               545550555560                                                                   ProGlySerLeuHisLeuLysAsnGlyProGluSerIleMetAsnSer                               565570575                                                                      ThrGluTyrProHisGlyGlySerGlnGlyGlnLeuLeuHisProGly                               580585590                                                                      AspHisLysAlaGlnAlaHisAsnLysAlaLeuProProGlyLeuSer                               595600605                                                                      LysGluGlnAlaProAspCysSerSerSerAspGlySerGluArgSer                               610615620                                                                      TrpMetAspAspTyrAspTyrValHisLeuGlnGlyLysGluGluPhe                               625630635640                                                                   GluArgGlnGlnLysGluLeuLeuGluLysGluAsnIleMetLysGln                               645650655                                                                      AsnLysMetGlnLeuGluHisHisGlnLeuSerGlnPheGlnLeuLeu                               660665670                                                                      GluGlnGluIleThrLysProValGluAsnAspIleSerLysTrpLys                               675680685                                                                      ProSerGlnSerLeuProThrThrAsnSerGlyValSerAlaGlnAsp                               690695700                                                                      ArgGlnLeuLeuCysPheTyrTyrAspGlnCysGluThrHisPheIle                               705710715720                                                                   SerLeuLeuAsnAlaIleAspAlaLeuPheSerCysValSerSerAla                               725730735                                                                      GlnProProArgIlePheValAlaHisSerLysPheValIleLeuSer                               740745750                                                                      AlaHisLysLeuValPheIleGlyAspThrLeuThrArgGlnValThr                               755760765                                                                      AlaGlnAspIleArgAsnLysValMetAsnSerSerAsnGlnLeuCys                               770775780                                                                      GluGlnLeuLysThrIleValMetAlaThrLysMetAlaAlaLeuHis                               785790795800                                                                   TyrProSerThrThrAlaLeuGlnGluMetValHisGlnValThrAsp                               805810815                                                                      LeuSerArgAsnAlaGlnLeuPheLysArgSerLeuLeuGluMetAla                               820825830                                                                      ThrPhe                                                                         (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 872 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: Not Relevant                                                 (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        MetLysTyrLeuAsnValLeuAlaLysAlaLeuTyrAspAsnValAla                               151015                                                                         GluSerProAspGluLeuSerPheArgLysGlyAspIleMetThrVal                               202530                                                                         GluArgAspThrGlnGlyLeuAspGlyTrpTrpLeuCysSerLeuHis                               354045                                                                         GlyArgGlnGlyIleValProGlyAsnArgLeuLysIleLeuValGly                               505560                                                                         MetTyrAspLysLysProAlaAlaProGlyProGlyProProAlaThr                               65707580                                                                       ProProGlnProGlnProSerLeuProGlnGlyValHisThrProVal                               859095                                                                         ProProAlaSerGlnTyrSerProMetLeuProThrAlaTyrGlnPro                               100105110                                                                      GlnProAspAsnValTyrLeuValProThrProSerLysThrGlnGln                               115120125                                                                      GlyLeuTyrGlnAlaProGlyAsnProGlnPheGlnSerProProAla                               130135140                                                                      LysGlnThrSerThrPheSerLysGlnThrProHisHisSerPhePro                               145150155160                                                                   SerProAlaThrAspLeuTyrGlnValProProGlyProGlySerPro                               165170175                                                                      AlaGlnAspIleTyrGlnValProProSerAlaGlyThrGlyHisAsp                               180185190                                                                      IleTyrGlnValProProSerLeuAspThrArgSerTrpGluGlyThr                               195200205                                                                      LysProProAlaLysValValValProThrArgValGlyGlnGlyTyr                               210215220                                                                      ValTyrGluAlaSerGlnAlaGluGlnAspGluTyrAspThrProArg                               225230235240                                                                   HisLeuLeuAlaProGlySerGlnAspIleTyrAspValProProVal                               245250255                                                                      ArgGlyLeuLeuProAsnGlnTyrGlyGlnGluValTyrAspThrPro                               260265270                                                                      ProMetAlaValLysGlyProAsnGlyArgAspProLeuLeuAspVal                               275280285                                                                      TyrAspValProProSerValGluLysGlyLeuProProSerAsnHis                               290295300                                                                      HisSerValTyrAspValProProSerValSerLysAspValProAsp                               305310315320                                                                   GlyProLeuLeuArgGluGluThrTyrAspValProProAlaPheAla                               325330335                                                                      LysProLysProPheAspProThrArgHisProLeuIleLeuAlaAla                               340345350                                                                      ProProProAspSerProProAlaGluAspValTyrAspValProPro                               355360365                                                                      ProAlaProAspLeuTyrAspValProProGlyLeuArgArgProGly                               370375380                                                                      ProGlyThrLeuTyrAspValProArgGluArgValLeuProProGlu                               385390395400                                                                   ValAlaAspGlySerValIleAspAspGlyValTyrAlaValProPro                               405410415                                                                      ProAlaGluArgGluAlaProThrAspGlyLysArgLeuSerAlaSer                               420425430                                                                      SerThrGlySerThrArgSerSerGlnSerAlaSerSerLeuGluVal                               435440445                                                                      ValValProGlyArgGluProLeuGluLeuGluValAlaValGluThr                               450455460                                                                      LeuAlaArgLeuGlnGlnGlyValSerThrThrValAlaHisLeuLeu                               465470475480                                                                   AspLeuValGlySerAlaSerGlyProGlyGlyTrpArgSerThrSer                               485490495                                                                      GluProGlnGluProProValGlnAspLeuLysAlaAlaValAlaAla                               500505510                                                                      ValHisGlyAlaValHisGluLeuLeuGluPheAlaArgSerAlaVal                               515520525                                                                      SerSerAlaThrHisThrSerAspArgThrLeuHisAlaLysLeuSer                               530535540                                                                      ArgGlnLeuGlnLysMetGluAspValTyrGlnThrLeuValValHis                               545550555560                                                                   GlyGlnValLeuAspSerGlyArgGlyGlyProGlyPheThrLeuAsp                               565570575                                                                      AspLeuAspThrLeuValAlaCysSerArgAlaValProGluAspAla                               580585590                                                                      LysGlnLeuAlaSerPheLeuHisGlyAsnAlaSerLeuLeuPheArg                               595600605                                                                      ArgThrLysAlaProGlyProGlyProGluGlySerSerSerLeuHis                               610615620                                                                      LeuAsnProThrAspLysAlaSerSerIleGlnSerArgProLeuPro                               625630635640                                                                   SerProProLysPheThrSerGlnAspSerProAspGlyGlnTyrGlu                               645650655                                                                      AsnSerGluGlyGlyTrpMetGluAspTyrAspTyrValHisLeuGln                               660665670                                                                      GlyLysGluGluPheGluLysThrGlnLysGluLeuLeuGluLysGly                               675680685                                                                      AsnIleValArgGlnGlyLysGlyGlnLeuGluLeuGlnGlnLeuLys                               690695700                                                                      GlnPheGluArgLeuGluGlnGluValSerArgProIleAspHisAsp                               705710715720                                                                   LeuAlaAsnTrpThrProAlaGlnProLeuValProGlyArgThrGly                               725730735                                                                      GlyLeuGlyProSerAspArgGlnLeuLeuLeuPheTyrLeuGluGln                               740745750                                                                      CysGluAlaAsnLeuThrThrLeuThrAspAlaValAspAlaPhePhe                               755760765                                                                      ThrAlaValAlaThrAsnGlnProProLysIlePheValAlaHisSer                               770775780                                                                      LysPheValIleLeuSerAlaHisLysLeuValPheIleGlyAspThr                               785790795800                                                                   LeuSerArgGlnAlaLysAlaAlaAspValArgSerLysValThrHis                               805810815                                                                      TyrSerAsnLeuLeuCysAspLeuLeuArgGlyIleValAlaThrThr                               820825830                                                                      LysAlaAlaAlaLeuGlnTyrProSerProSerAlaAlaGlnAspMet                               835840845                                                                      ValAspArgValLysGluLeuGlyHisSerThrGlnGlnPheArgArg                               850855860                                                                      ValLeuGlyGlnLeuAlaAlaAla                                                       865870                                                                         (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 78 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: Not Relevant                                                 (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (v) FRAGMENT TYPE: C-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        LeuSerGlnPheGlnLeuLeuGluGlnGluIleThrLysProValGlu                               151015                                                                         AsnAspIleSerLysTrpLysProSerGlnSerLeuProThrThrAsn                               202530                                                                         AsnSerValGlyAlaGlnAspArgGlnLeuLeuCysPheTyrTyrAsp                               354045                                                                         GlnCysGluThrHisPheIleSerLeuLeuAsnAlaIleAspAlaLeu                               505560                                                                         PheSerCysValSerSerAlaGlnProProArgIlePheVal                                     657075                                                                         __________________________________________________________________________ 

What is claimed is:
 1. An isolated double-stranded nucleic acid molecule which specifically hybridizes with SEQ. ID No. 1, said nucleic acid molecule comprising an open reading frame encoding a human signal mediator protein between about 795 and about 875 amino acids in length, said encoded protein comprising an amino-terminal SH3 domain, an internal domain that includes a multiplicity of SH2 binding motifs, said SH2 binding motifs being encoded by consensus nucleic acid sequences and a carboxy-terminal effector domain, said effector domain, when expressed in Saccharomyces cerevisiae, inducing pseudohyphal budding in said Saccharomyces cerevisiae under low nitrogen culture conditions.
 2. The nucleic acid molecule of claim 1, which is DNA.
 3. The DNA molecule of claim 2, which is a cDNA comprising a sequence approximately 3.7 kilobase pairs in length that encodes said signal mediator protein.
 4. The DNA molecule of claim 2, which is a gone comprising exons, the exons of said gene specifically hybridizing with the nucleic acids of SEQ. ID No. 1, and said exons encoding said signal mediator protein.
 5. An isolated RNA molecule transcribed from the double-stranded nucleic acid molecule of claim
 1. 6. The nucleic acid molecule of claim 1, wherein said open reading frame encodes a human Signal mediator protein comprising an amino acid sequence selected from the group consisting of an amino acid sequence encoded by natural allelic variants of said open reading frame and and SEQ. I.D. No.
 2. 7. An isolated nucleic acid molecule comprising an open reading frame encoding a mammalian signal mediator protein, wherein the sequence of said protein is SEQ. I.D. No.
 2. 8. The nucleic acid molecule of claim 7, which comprises SEQ I.D. No.
 1. 9. An isolated nucleic acid molecule comprising a sequence selected from the group consisting of:a) SEQ, I.D. No. 1; b) a sequence which specifically hybridizes with SEQ. I.D. No. 1 c) a sequence encoding a polypeptide of SEQ. I.D No. 2; and d) a nucleic acid sequence encoding a carboxy terminal effector domain of a signal mediator protein having an amino acid sequence corresponding to amino acids 626-834 of SEQ. ID No.
 2. 10. An isolated double-stranded nucleic acid molecule which specifically hybridizes with SEQ. ID No. 1, said nucleic acid molecule comprising an open reading frame encoding a mouse signal mediator protein, said protein further comprising an amino-terminal SH3 domain, an internal domain that includes a multiplicity of SH2 binding motifs, said SH2 binding motifs being encoded by consensus nucleic acid sequences and a carboxy-terminal effector domain, said effector domain, when expressed in Saccharomyces cerevisiae, inducing pseudohyphal budding in said Saccharomyces cerevisiae under low nitrogen culture conditions.
 11. The nucleic acid molecule of claim 10, which is DNA.
 12. The DNA molecule of claim 11, which is a cDNA.
 13. The DNA molecule of claim 11, which is a gene comprising exons, the exons of said gene specifically hybridizing with nucleic acid sequences of SEQ. ID No. 1, and said exons encoding said mouse signal mediator protein.
 14. An isolated RNA molecule transcribed from the double-stranded nucleic acid molecule of claim
 10. 15. An oligonucleotide between about 10 and about 100 nucleotides in length, which specifically hybridizes with a nucleotide sequence encoding amino acids of SEQ. I.D. No.
 4. 16. An isolated nucleic acid molecule according to claim 1 encoding a human signal mediator protein of about 834 amino acids in length.
 17. An oligonucleotide between about 10 and about 100 nucleotides in length, which specifically hybridizes with a sequence in the nucleic acid molecule of claim 1, said sequence encoding a serine-rich region of said signal mediator protein. 